While the traditional focus of basic and clinical cancer immunology research has been on MHC class I-restricted cytolytic CD8+ T lymphocytes, our laboratory has innovated investigations into the role of MHC class II-restricted CD4+ T cells in human antitumor immunity. CD4+ cells can be crucial for initiating, orchestrating and maintaining antitumor immune responses in animal models. This laboratory has been on the forefront of developing new technologies for the biochemical and molecular identification of tumor-associated proteins recognized by CD4+ T cells, including protein purification and cDNA library screening approaches targeting the MHC class II Ag processing/presentation pathway. Current projects include: 1) Clinical investigations using the melanoma-associated Ag tyrosinase (Ty) as an immunogen. We previously demonstrated that Ty, an enzyme catalyzing melanogenesis and expressed in metastatic melanoma lesions, could be targeted by both CD4+ and CD8+ T cells. Poxvirus vectors encoding Ty are now being used, in conjunction with systemic interleukin-2, to treat patients with stage IV metastatic melanoma. As part of this project, a major effort has been made to develop new in vitro methods for monitoring immune reactivities to whole proteins such as Ty. Recombinant adenovirus was used to express Ty in autologous dendritic cells, coupled with real time quantitative RT-PCR to assess cytokine mRNA levels in PBL from vaccinated patients after transient in vitro Ag stimulation. A current project aims to develop a lentiviral vector for this purpose, which can be expressed in several different kinds of Ag presenting cells without encoding extraneous proteins. Such methods may also be useful for the efficient evaluation of "candidate" tumor Ag identified through genomic or proteomic screening. 2) Evaluation of novel tumor Ag for clinical application. Following the recent finding that a single mutation in the signaling molecule BRAF is associated with >60% of melanomas, our laboratory has investigated whether mutated BRAF can be recognized by CD4+ T cells from melanoma patients. T cells specific for a mutated peptide, restricted by 4 different MHC class II alleles, have been generated and are currently under study for their ability to recognize tumor cells harboring the mutation. Another tumor Ag identified in our lab through molecular cloning, neo-poly(A) polymerase, is widely expressed in tumors including melanomas, prostate cancers and colon cancers. In this case, CD8+ T cells recognizing HLA-A1 and -A2 restricted neo-PAP peptides have been raised, and are now being tested for tumor cell recognition. 3) Characterization of a T cell receptor specific for a shared prostate/colon cancer Ag. Our studies of T cell reactivity to human prostate cancer identified a "nonclassical" CD8+ T cell capable of recognizing a shared prostate/colon cancer Ag but not restricted by conventional MHC molecules. Limited quantities of the reactive T cell clone precluded complete analysis, so the TCRab has been cloned into a retroviral vector for expression in PBL. If functional, this TCR will have potential clinical utility and may allow identification of a tumor Ag with wide clinical applicability.